The objective of this application is to determine the structure and kinetics of the reaction intermediates in the reduction of 02 to H20 by cytochrome oxidase so as to delineate the catalytic mechanism of this enzyme. In the proposed research a comprehensive approach will be taken in which the same intermediates will be studied by several physical techniques and will be generated in different ways. To do this we will combine the resources and skills of our research groups at the University of Pennsylvania and at AT&T Bell Laboratories. We plan to use optical absorption, resonance Raman scattering and electron paramagnetic resonance to study reaction intermediates from three different types of enzyme preparations - the Hartzell- Beinert, Yonetani, and Yoshikawa-Caughey preparations. To study the short lived intermediates we will make flow/flash/probe measurements with a time resolution of 1 musec. The primary goal of this phase of the work will be to identify and characterize the first intermediate involving the binding of 02 to cytochrome oxidase. We wish to determine if properties of the initial bound complex can account for the rapid 02 reduction which occurs. Once the initial intermediate is characterize we will proceed to characterize other early intermediates. The longer time intermediates (is greater than 10 msec) will be studied by rapid mixing of the fully reduced cytochrome oxidase with an oxygen saturated solution and following the reaction to the resting state. The initial goal of the rapid mixing studies will be to characterize "pulsed" cytochrome oxidase. Recent studies of this species have not yielded a consistent set of properties and consequently there is no agreement on the properties or the structure of this important intermediate. In additional work we plan to study the other long-lived intermediates in the relaxation of the enzyme from the oxygen-bound reduced state to the resting state. Low temperature intermediates will be studied by the triple trapping technique. The objective of the triple trapping studies will be to determine if the relaxation pathways (and the properties of the intermediates) are the same at low temperature as they are at room temperature. By pursuing this comprehensive approach it is expected that the many inconsistencies in the literature will be resolved and that a thorough understanding of the catalytic mechanism of cytochrome oxidase will be achieved.